Recombinant Monosiga brevicollis DDRGK domain-containing protein 1 (22450)

Basic Research Questions

• What is DDRGK domain-containing protein 1 and what is its functional significance in Monosiga brevicollis?

DDRGK domain-containing protein 1 (DDRGK1) is a critical component of the ufmylation system, a post-translational modification pathway that involves the covalent attachment of UFM1 (Ubiquitin-fold modifier 1) to substrate proteins. In Monosiga brevicollis, DDRGK1 likely serves as a key regulator of endoplasmic reticulum (ER) homeostasis and cellular stress responses, similar to its function in metazoans .

Studies in other model organisms have demonstrated that DDRGK1 has a dual role in autophagy regulation: it inhibits autophagy induction by supporting mTOR signaling while facilitating autophagosome-lysosome fusion for proper degradation . This protein's presence in M. brevicollis, a unicellular organism that is the closest living relative to metazoans, suggests that the ufmylation pathway predates multicellularity and may have played a fundamental role in early eukaryotic cellular processes .

Experimental approaches to characterize M. brevicollis DDRGK1 typically involve:

• Gene expression analysis using RT-PCR or RNA-seq

• Protein localization studies using immunofluorescence

• Functional assays measuring impacts on ER stress and autophagy markers

• What evolutionary insights can be gained from studying DDRGK1 in Monosiga brevicollis?

Monosiga brevicollis occupies a unique evolutionary position as the closest unicellular relative to metazoans, making its molecular mechanisms particularly valuable for understanding the transition to multicellularity. The genome of M. brevicollis contains approximately 9,200 intron-rich genes, including many that encode signaling and adhesion domains previously thought to be exclusive to multicellular animals .

Studying DDRGK1 in this choanoflagellate provides several evolutionary insights:

• It helps establish when the ufmylation system first emerged in the eukaryotic lineage

• It allows researchers to track domain shuffling events that occurred after the divergence of choanoflagellates and metazoans

• It illuminates how protein interaction networks involving DDRGK1 evolved during the transition to multicellularity

Comparative genomic analyses have revealed that while M. brevicollis possesses many protein domains found in metazoans, the physical linkages between these domains often differ significantly, suggesting extensive domain shuffling during evolution . For DDRGK1 specifically, this context helps researchers understand how the ufmylation system was potentially co-opted or modified during the evolution of multicellular organisms.

• How is recombinant M. brevicollis DDRGK1 typically expressed and purified for research applications?

Expression and purification of recombinant M. brevicollis DDRGK1 typically follows established protocols for challenging eukaryotic proteins, with several modifications to address the specific characteristics of choanoflagellate proteins:

Expression System Selection:
The bacterial expression system (E. coli) is commonly used for initial attempts, but researchers often encounter solubility issues due to the complex nature of DDRGK1. For functional studies, eukaryotic expression systems such as insect cells (Sf9 or Hi5) or mammalian cells (HEK293) are preferable to ensure proper folding and post-translational modifications.

Optimization Protocol:

• Clone the M. brevicollis DDRGK1 gene (UniProt ID: MONBRDRAFT_22450) into an appropriate expression vector with an affinity tag (typically His6 or GST)

• Transform/transfect the host system and optimize expression conditions (temperature, induction time, media composition)

• Lyse cells using methods that preserve protein structure (e.g., gentle detergents for membrane-associated fractions)

• Purify using affinity chromatography followed by size exclusion chromatography

Key Challenges:

• The high GC content (55%) of the M. brevicollis genome can cause expression issues in some hosts

• DDRGK1 may require specific chaperones or co-factors for proper folding

• As a component of the ufmylation system, DDRGK1 likely participates in protein complexes and may be unstable in isolation

Similar approaches have been successfully applied to other M. brevicollis proteins, such as the tyrosine kinase HMTK1, which was expressed, purified, and demonstrated to possess enzymatic activity in vitro .

Advanced Research Questions

• What is the relationship between M. brevicollis DDRGK1 and the ufmylation pathway in the context of evolutionary cell biology?

The ufmylation pathway appears to be conserved across eukaryotes with secondary loss in some lineages, as indicated by phylogenomic analyses . In M. brevicollis, DDRGK1 likely functions as part of this ancient post-translational modification system that predates the emergence of multicellularity.

Recent research suggests a crucial connection between the ufmylation system and selective autophagy pathways, specifically ER-phagy (the selective degradation of the endoplasmic reticulum via autophagy) . The mechanistic details uncovered in other model organisms indicate that:

• Ribosome-ER collisions trigger ribosomal UFMylation

• This modification activates C53-mediated autophagy

• The autophagy machinery then clears toxic incomplete polypeptides

• DDRGK1 regulates UFMylation activity when bound to the UFL1-DDRGK1 complex

M. brevicollis DDRGK1 likely participates in this cellular quality control mechanism, representing one of the earliest examples of this pathway in evolutionary history. This is particularly significant because it suggests that sophisticated cellular quality control systems were present in the last common ancestor of choanoflagellates and metazoans, potentially facilitating the evolution of complex multicellular organisms.

The study of M. brevicollis DDRGK1 provides a unique opportunity to understand the ancestral functions of the ufmylation pathway before it was potentially adapted for specialized roles in multicellular organisms.

• What methodological approaches would be most effective for characterizing protein-protein interactions involving M. brevicollis DDRGK1?

Characterizing the interactome of M. brevicollis DDRGK1 requires specialized approaches that account for both its evolutionary position and functional context:

Recommended Methods and Their Applications:

Based on studies of other M. brevicollis proteins, the PDZ domain-containing proteins in particular, a combination of structural characterization and binding affinity measurements has proven effective . For DDRGK1, researchers should focus on identifying interactions with:

• Components of the UFMylation machinery (UFM1, UFL1)

• Potential substrate proteins

• Components of the autophagy pathway

A particularly informative approach would be to compare the interactome of M. brevicollis DDRGK1 with its metazoan homologs to identify conserved and divergent interaction partners, providing insights into the evolution of this important cellular pathway.

• How does the domain architecture of M. brevicollis DDRGK1 compare with its metazoan counterparts, and what does this reveal about domain shuffling during evolution?

The domain architecture of proteins in M. brevicollis often differs significantly from their metazoan counterparts despite containing similar functional domains. This pattern is consistent with the hypothesis that extensive domain shuffling occurred following the divergence of choanoflagellates and metazoans .

For signaling domains involved in phosphotyrosine signaling, more than half of the observed pairwise domain combinations in M. brevicollis are distinct from those seen in metazoans . This divergence in domain architecture is particularly pronounced compared to other signaling systems (such as phosphoserine/threonine, Ras-GTP, and Rho-GTP signaling), which show greater conservation of domain combinations .

Comparative Domain Analysis of DDRGK1:

While the search results don't provide the specific domain architecture of M. brevicollis DDRGK1, the general pattern observed in other proteins suggests that it may contain the core DDRGK domain with different flanking domains or in different contexts compared to metazoan homologs. This would be consistent with the observation that M. brevicollis proteins display "a wealth of combinations of known signaling domains" .

Understanding these differences can provide insights into:

• The minimal functional requirements for DDRGK1 activity

• How new functions may have evolved through domain shuffling

• The ancestral state of the protein before the emergence of multicellularity

• What functional assays can be employed to assess the role of recombinant M. brevicollis DDRGK1 in autophagy regulation?

Based on studies of DDRGK1 in other organisms, several functional assays can be adapted to assess the role of recombinant M. brevicollis DDRGK1 in autophagy regulation:

In Vitro Assays:

• UFMylation Activity Assay: Monitor the transfer of UFM1 to substrate proteins in the presence of recombinant M. brevicollis DDRGK1, UFL1, and other components of the ufmylation machinery using Western blotting or mass spectrometry.

• Protein-Protein Interaction Assays: Use pull-down assays, surface plasmon resonance, or isothermal titration calorimetry to characterize interactions between recombinant DDRGK1 and components of the autophagy machinery.

Cell-Based Assays:

• Complementation Experiments: Express M. brevicollis DDRGK1 in DDRGK1-deficient mammalian cells (such as the 4-OHT-treated MEFs described in search result ) and assess rescue of autophagy defects.

• Autophagy Flux Assessment: Monitor autophagy markers such as LC3-II/LC3-I ratio, p62 levels, and autophagosome-lysosome fusion in the presence of wild-type or mutant M. brevicollis DDRGK1.

• Lysosomal Function Tests: Assess lysosomal pH, Cathepsin D expression, and v-ATPase accumulation, as DDRGK1 loss has been correlated with suppressed lysosomal function .

Experimental Design Considerations:

These assays would help determine whether the dual role of DDRGK1 in autophagy regulation (promoting induction while also facilitating degradation) is conserved in choanoflagellates, providing insights into the evolution of this regulatory mechanism.

• How might structural studies of M. brevicollis DDRGK1 contribute to our understanding of protein evolution and function?

Structural studies of M. brevicollis DDRGK1 would provide valuable insights into both its molecular function and evolutionary history. Previous structural studies of other M. brevicollis proteins, such as the PDZ domains and the kinase domain of receptor tyrosine kinase C8 (RTKC8), have yielded important evolutionary insights .

Potential Contributions of Structural Studies:

• Ancestral Structure Reconstruction: The structure of M. brevicollis DDRGK1 could represent an ancestral form of the protein, predating the divergence of choanoflagellates and metazoans. This would provide a "fossil record" of protein structure before the evolution of multicellularity.

• Structure-Function Relationships: Comparing the structure of M. brevicollis DDRGK1 with its metazoan counterparts could reveal conserved functional regions versus areas that have undergone adaptive evolution. This would help define the core functional elements of DDRGK1 that have been maintained throughout evolution.

• Interaction Interfaces: Structural studies, particularly co-crystal structures with binding partners, could identify the molecular determinants of specific protein-protein interactions in the ufmylation pathway. For example, the crystal structure of the RTKC8 kinase domain revealed details about its active conformation and substrate binding .

• Domain Organization: The three-dimensional arrangement of domains in M. brevicollis DDRGK1 could provide insights into how domain shuffling affects protein function. This is particularly relevant given the extensive domain shuffling observed in choanoflagellate proteins compared to their metazoan counterparts .

Methodological Approaches:

• X-ray crystallography of the isolated DDRGK domain or full-length protein

• Cryo-electron microscopy of DDRGK1 in complex with interaction partners

• NMR spectroscopy for dynamic regions or smaller domains

• Computational modeling and molecular dynamics simulations

Such structural studies would complement the genomic and biochemical analyses of M. brevicollis DDRGK1, providing a more complete picture of this protein's role in early eukaryotic evolution.

Technical and Methodological Questions

• What are the key considerations for designing expression constructs to optimize the solubility and activity of recombinant M. brevicollis DDRGK1?

Designing expression constructs for M. brevicollis DDRGK1 requires careful consideration of several factors to optimize protein solubility, stability, and activity:

Domain Boundary Optimization:

• Perform bioinformatic analyses (e.g., SMART, Pfam, InterPro) to accurately identify domain boundaries

• Consider constructing multiple variants with different N- and C-terminal boundaries

• Evaluate flexible regions that might interfere with proper folding or crystallization using disorder prediction tools (PONDR, IUPred)

Codon Optimization:

• The M. brevicollis genome has a GC content of approximately 55% , which may necessitate codon optimization for expression in common host systems

• Design synthetic genes with codons optimized for the chosen expression system while avoiding rare codons that could impede translation

Fusion Tags and Solubility Enhancers:

• N-terminal tags: His6, GST, MBP, SUMO (particularly effective for enhancing solubility)

• C-terminal tags: Consider smaller tags (e.g., FLAG, Strep-tag II) to minimize interference with DDRGK domain function

• Include TEV or PreScission protease cleavage sites for tag removal

Expression System Selection:

• Bacterial systems: BL21(DE3), Rosetta(DE3), or SHuffle strains for proteins requiring disulfide bond formation

• Eukaryotic systems: Consider insect cells (Sf9, Hi5) or mammalian cells (HEK293) for complex proteins requiring post-translational modifications

Based on experiences with other M. brevicollis proteins, such as HMTK1 and RTKC8 , prokaryotic expression systems can successfully produce functional proteins, but optimization of expression conditions (temperature, induction time, media composition) is critical.

• What experimental approaches can be used to investigate the evolutionary relationship between UFMylation and autophagy using M. brevicollis as a model organism?

Investigating the evolutionary relationship between UFMylation and autophagy using M. brevicollis as a model organism requires a multifaceted approach that integrates phylogenetic, functional, and comparative analyses:

Phylogenomic Analysis:

• Conduct comprehensive phylogenetic analysis of all components of the UFMylation and autophagy pathways across diverse eukaryotes

• Map the presence/absence patterns of key components to identify co-evolutionary relationships

• Perform ancestral state reconstruction to determine which components were present in the last common ancestor of choanoflagellates and metazoans

This approach has revealed that UFMylation is conserved across eukaryotes with secondary loss in some lineages , providing a foundation for more detailed functional studies.

Functional Characterization in M. brevicollis:

Cross-Species Functional Studies:

• Express M. brevicollis DDRGK1 in mammalian or yeast cells lacking the endogenous protein

• Assess the ability of the choanoflagellate protein to complement loss-of-function phenotypes

• Compare the interactomes of M. brevicollis and metazoan DDRGK1 proteins to identify conserved and divergent interaction partners

Structural Biology:

• Determine structures of key protein complexes involved in both pathways

• Focus on interaction interfaces between UFMylation components (DDRGK1-UFL1) and autophagy machinery

• Compare these structures with those from metazoans to identify evolutionary changes

These approaches would help determine whether the connection between UFMylation and autophagy observed in metazoans (specifically, the role of UFMylation in ER-phagy and C53-mediated autophagy ) is an ancestral feature present in choanoflagellates or a derived feature that evolved in the metazoan lineage.